Methods, kits, and compositions for enhancing cellular therapy

ABSTRACT

Cell-based compositions and methods for targeting and treating human diseases, including cancers and infectious diseases, are provided, wherein exogenous intracellular sarcosine is used for improved delivery of the composition.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119 of U.S.provisional application 62/311,441, filed Mar. 22, 2016, the entirecontents of which is incorporated herein by reference.

BACKGROUND

Immunotherapy is an approach to the treatment of cancer. In thetreatment of brain tumors, one treatment approach is to provide patientswith a vaccine of their own dendritic cells that are loaded with aparticular brain tumor antigen. These dendritic cells migrate from thesite of injection to a draining lymph node where they stimulate T cellsto generate an immune response against the target antigen. The T cellstravel to the brain tumor to identify cells expressing the targetantigen, resulting in tumor killing.

Enhancing migration of dendritic cells (DCs) to the local lymph node canbe important for the efficacy of this treatment strategy, and braintumor outcomes in humans correlate with dendritic cell (DC) migration(Mitchell et al., Nature, 2015, 519 (19), incorporated herein byreference, in its entirety). Mitchell et al. (2015) show that byenhancing DC migration using a tetanus toxoid recall response, patientswith malignant brain tumors experience improved survival. Enhancingmigration of these and/or other cells, e.g., T cells used in adoptivecell therapy or hematopoietic stem cells used in hematopoietic stem celltransplantation therapy, is important to improve immune responses tovaccination and to improve targeting of disease cells.

SUMMARY

The current disclosure demonstrates the novel finding that intracellularloading of DCs with a common metabolite called sarcosine significantlyenhances DC migration. The cells were electroporated with sarcosine andcultured in sarcosine containing medium and then used to vaccinate mice.After a period of time, the lymph nodes were harvested and DC migrationevaluated by using flow cytometry to evaluate the presence of theinjected DCs. The data shows that the combination of sarcosine loadedDCs and the tetanus toxoid strategy results in a synergistic effect onmigration of the cells to local lymph nodes. This novel discovery isimportant because enhancing migration of dendritic cells can improve theimmune response to vaccination and improve killing of disease cells,e.g., malignant brain tumor cells, thereby increasing treatmentresponse, improving patient survival and potentially achieving a cure.The sarcosine method described herein is novel and can be applied toother cells such as T cells in an adoptive immunotherapy strategy orhematopoietic cells in hematopoietic stem cell transplantation therapy.

According to one aspect of the invention, a composition is provided. Thecomposition contains an antigen presenting cell (APC), wherein the cellcomprises a receptor for presenting antigen and antigen bound to thereceptor, and wherein the cell contains above physiological levels ofsarcosine. Other cells that can be used as antigen presenting cellsinclude T cells, macrophages, hematopoietic stem cells, B cells, antigenpresenting cells derived from induced pluripotent stem cells (iPSCs),and genetically modified autologous cells (fibroblasts, tumor cells,mesenchymal stem cells).

According to another aspect of the invention, a composition is provided.The composition contains an antigen presenting cell (APC), wherein thecell comprises a receptor for presenting antigen and antigen bound tothe receptor, and wherein the cell is electroporated with sarcosineand/or the cell is cultured in the presence of media containingsarcosine.

According to another aspect of the invention, a method is provided forpreparing a cell-based pharmaceutical preparation. The method involvesproviding an antigen presenting cell comprising a receptor forpresenting antigen and contacting the cell with a solution containingsarcosine.

According to a further aspect of the invention, a method is provided fortreating a disease in a subject. The method involves administering tothe subject an antigen presenting cell (APC), wherein the cell comprisesa receptor for presenting antigen and antigen bound to the receptor andwherein the cell contains above physiological levels of sarcosine. Inthis aspect of the invention the subject may be treated using a strategyfor enhancing migration of dendritic cells (DCs) to the local lymphnode, using, for example, a tetanus toxoid administration/primingstrategy as is described in Mitchell et al., Nature, 2015, 519 (19),herein incorporated by reference, in its entirety. In some aspects ofthe invention the subject may be treated using a strategy for enhancingmigration of dendritic cells (DCs) to the local lymph node using othercellular therapies, for example, Provenge (Dendreon), DCVax (NorthwestBiotherapeutics), AGS vaccines (Argos Therapeutics), or CAR T cells(Juno Therapeutics, Novartis, Kite Pharma, and Cellestis).

According to another aspect of the invention, a cell culture compositionis provided. The composition includes a liquid medium containingsarcosine in concentrations above physiological levels, and an antigenpresenting cell (APC). In embodiments, the antigen presenting cellcomprises a receptor for presenting antigen. In embodiments, the antigenpresenting cell includes antigen bound to the receptor. In embodiments,the liquid medium contains the antigen.

In any of the foregoing embodiments, the sarcosine in the cell can bepresent in an amount of at least 0.1 pg, or in an amount between about0.1 pg and 15 pg, between about 0.2 pg and 10 pg, between about 0.3 pgand 5 pg, or between about 0.5 pg and 3.5 pg of sarcosine. Inembodiments, the cell can contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, or3.5 pg sarcosine. In any of the foregoing embodiments, the concentrationof sarcosine in the media can be about 6 mM to about 1 molar, forexample, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100 mM.

In any of the foregoing embodiments, the APC can be a mammalian cell. Inany of the foregoing embodiments, the APC can be a human cell. In any ofthe foregoing embodiments, the APC can be a dendritic cell (DC),including but not limited to a mammalian or a human dendritic cell. Inany of the foregoing embodiments, the antigen can be adisease-associated antigen.

In any of the foregoing embodiments, the antigen can be a tumor antigen.In any of the foregoing embodiments, the antigen can be a brain tumorantigen. In any of the foregoing embodiments, the antigen can be aglioblastoma antigen. In any of the foregoing embodiments, the antigencan be derived from total tumor RNA.

In any of the foregoing embodiments, the tumor antigen can be an antigenpresented on or within brain cancer cells, e.g., glioblastoma, MAGE-1,AIM-2, gp100, or HER2.

In any of the foregoing embodiments, the antigen can be an infectiousdisease antigen. In any of the foregoing embodiments, the antigen can bea cytomegalovirus (CMV) antigen.

In any of the foregoing embodiments, the antigen can be a conservedmutation antigen, e.g., IDH1, K27M, BRAF V600E, or EGFRvIII mutation.

In any of the foregoing embodiments, the antigen can be apatient-specific mutation antigen.

In any of the foregoing embodiments, the antigen can be an autoimmunedisease antigen. In any of the foregoing embodiments, the antigen can bea self-antigen.

In any of the foregoing embodiments, the antigen can be presented to theAPC by: pulsing the APC (e.g., co-culture or electroporation); fusingcell components with disease tissue components, e.g., tumor components;RNA transfection; viral gene transduction; or DNA transfer.

In any of the foregoing embodiments, the antigen can be a short peptide,a long peptide, mRNA, DNA, recombinant protein, tumor lysate mixture, orcarbohydrate antigen expressed within a cancer cell or infected cell.

In any of the foregoing embodiments, the antigen presenting cell can besubstituted with other cells such as T cells (for adoptive immunotherapystrategies) or hematopoietic cells (for hematopoietic stem celltransplantation therapy).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amounts (pg) of sarcosine per cell after differenttreatment conditions. Conditions: control cells (no sarcosine treatment)(column 1), 6 mM sarcosine in cell culture medium plus electroporationwith 6 mM sarcosine prior to culture (column 2), 20 mM sarcosine in cellculture medium plus electroporation with 0.5 M sarcosine prior toculture (column 3).

FIG. 2 shows in vitro trans-well cell migration assay of DCs underdifferent cellular conditions. Conditions: DCs alone (column 1), DCsloaded with sarcosine (column 2), DCs plus addition of cytokines (column3), sarcosine loaded DCs plus addition of cytokines (column 4).Cytokines used in assay (columns 3 and 4) are CCL-19 and CCL-21 at aconcentration of 250 ng/ml for each of the cytokines.

FIG. 3 shows the percentage of cells under different vaccine conditionsthat migrate to the local lymph node in vaccinated mice. PKH labeled DCs(PKH is a red fluorescent protein) were used for DC vaccination.Td=tetanus/diphtheria toxoid. Vaccine conditions: vaccination with PKHDCs (no tetanus toxoid priming) (column 1), vaccination with sarcosineloaded PKH (no tetanus toxoid priming) (column 2), tetanus toxoidpriming and vaccination with PKH DCs (column 3), tetanus toxoid primingand vaccination with sarcosine loaded PKH DCs (column 4).

FIG. 4 shows NMR results of sarcosine loaded DCs in a serial dilutionassay. Cells loaded with sarcosine clearly show two strong signals atabout 3.6-ppm and 2.6-ppm on the chemical shift axis that represent the—CH2— and N-CH3 of sarcosine, respectively. Loaded DCs can be traced to5% dilution.

FIG. 5 shows amounts (pg) of sarcosine per cell after differenttreatment conditions. Conditions: control cells (no sarcosine treatment)(column 1), 20 mM sarcosine in cell culture medium (column 2), 20 mMsarcosine in cell culture medium plus electroporation with 0.5 Msarcosine (column 3), 20 mM sarcosine in cell culture medium pluselectroporation with siRNA against SARDH (column 4).

DETAILED DESCRIPTION

The following detailed description is made by way of illustration ofcertain aspects of the disclosure. It is to be understood that otheraspects are contemplated and may be made without departing from thescope or spirit of the present disclosure. The following detaileddescription, therefore, is not to be taken in a limiting sense.Scientific and technical terms used herein have meanings commonly usedin the art unless otherwise specified. The definitions provided hereinare to facilitate understanding of certain terms used frequently hereinand are not meant to limit the scope of the present disclosure. Thesingular forms “a”, “an”, and “the” encompass the plural, unless thecontent clearly dictates otherwise. The term “or” is generally employedin its sense including “and/or” unless the content clearly dictatesotherwise. When referring to sarcosine, the terms “loaded”, “loading”,and/or “load” may be synonymous with the terms “provided”, “providing”,and/or “provide” and with the terms “treated”, “treating”, and/or“treat”. The terms “levels” of sarcosine and “amounts” of sarcosine maybe synonymous, as used herein.

Antigen and antigen presenting cell (APC). An antigen is a molecule thatserves as a ligand for receptors of immune cells, including leukocytes,such as T cells. An antigen presenting cell (APC) is a cell thatdisplays or presents on its surface an antigen, e.g., a foreign antigen.This is referred to as antigen presentation. APCs include macrophages, Blymphocytes, and dendritic cells (DCs). In some embodiments, an APC,e.g., a dendritic cell, is obtained from a subject, e.g., a humansubject. In some embodiments, the cell is then treated ex vivo andthereafter, administered to the subject.

Antigen presentation. Antigens may be bound to receptors on APCs,through various methods. In some embodiments, antigens are presented oncell receptors or allowed to bind cell receptors by: pulsing the cellwith antigen, e.g., co-culture or electroporation; fusing cellcomponents with disease tissue components, e.g., tumor components; RNAtransfection; viral gene transduction; or DNA transfer.

Sarcosine. Sarcosine, also known as N-methylglycine, is a metabolitethat can be found in nature. The sarcosine metabolite is synthesized bythe glycine N-methyltransferase (GMNT) enzyme which uses a methyl groupfrom S-adenosyl methionine to donate to glycine, creating sarcosine andS-adenosylhomocysteine. It can be metabolized by either the sarcosinedehydrogenase (SARDH) enzyme or pipecolic acid oxidase (PIPDX), theformer of which is highly expressed in the liver but not brain andconverts sarcosine into glycine. Sarcosine is an intermediate product inthe metabolism of choline to glycine.

Serum sarcosine concentrations independent of supplementation have beennoted to be, e.g., 102.3 ng/mL and 80.8 ng/mL in men and women,respectively. Mean urine concentrations of sarcosine have been noted tobe, e.g., 138.5 ng/mL and 94.8 ng/mL in men and women, respectively(Meyer T E, et al., “A reproducible and high-throughput HPLC/MS methodto separate sarcosine from α- and β-alanine and to quantify sarcosine inhuman serum and urine”, Anal Chem., 2011). Intracellular concentrationsof sarcosine have been found to be lower than serum and urineconcentrations. For example, it is found in the instant disclosure thatthe intracellular level of sarcosine in a dendritic cell (DC) is0.13±0.06 pg/cell. Sarcosine, as in the instant disclosure, can bemeasured intracellularly using a commercially available Sarcosine Assaykit from Sigma Aldrich®. The kit allows sarcosine quantification usingan enzymatic reaction and colorimetric analysis. A known number of DCsare lysed using the assay buffer and centrifuged at high speed. Thesupernatant is then collected and the amount of sarcosine is measuredand expressed as amount of sarcosine/cell.

In some embodiments, sarcosine is provided to cells, e.g., APCs, toproduce in the cells amounts of sarcosine above physiological levelsnormally found in such cells. In some embodiments, sarcosine is loadedin or provided to the cell. In some embodiments, sarcosine is loaded inor provided to the cell by adding sarcosine to cell culture medium andculturing the cells, wherein the amount of sarcosine added to the mediumis above physiological levels found in medium normally surroundingtissue. In some embodiments, exogenous sarcosine is loaded in orprovided to the cell by electroporation, wherein the amount of sarcosineloaded or provided is an amount needed to cause the cell to containabove physiological levels, i.e., above levels of sarcosine normally ortypically present in the cell. In some embodiments, sarcosine is loadedin and/or provided to the cell both in the cell culture medium and byelectroporation. In some embodiments, the methods used to providesarcosine to APCs may be applied to other cell types, includingsplenocytes, hematopoietic stem cells (e.g., for use in hematopoieticstem cell transplantation), or T cells (e.g., for use in adoptivecellular therapy). In some embodiments, cells are treated with (loadedor provided with) sarcosine to enhance migration of thesarcosine-treated cell to a disease cell, e.g., cancer/tumor cell.Non-limiting examples of loading sarcosine in or providing sarcosine toa cell are provided herein, in the Examples.

In some embodiments, antigens may be presented on the cell, e.g., APC,e.g., dendritic cell. Antigens that bind to a receptor on the cellinclude, e.g., self-antigens, tumor antigens, cytomegalovirus (CMV)antigens, antigens derived from total tumor RNA, disease-associatedantigens, tumor-associated antigens (TAAs), tumor-specific antigens(TSAs), conserved mutation antigens, and patient-specific mutationantigens. The invention embraces antigen presenting cells loaded withany one of these antigens types.

Self-antigen. A self-antigen is an antigen that originates from within abody. Self-antigens may be expressed by tumor cells as well as somenormal cells. In some embodiments, tumor cells express self-antigens atan expression level higher than the expression level at which a normalcell expresses the same self-antigen. That is, the self-antigenexpressed by a tumor cell is overexpressed in the tumor, but present inthe normal cell.

Disease-associated antigen. A disease-associated antigen is one that isexpressed by the disease causing agent (e.g., an antigen of aninfectious disease agent) and/or is present in higher amounts ondiseased cells than on normal cells of the same type (e.g., certaintumor antigens).

Tumor antigen. A tumor antigen is an antigen expressed by tumor cellsbut (i) not on normal cells of the same type or (ii) in materiallyhigher amounts than expressed on normal tissue of the same type, suchthat the antigen is useful as an immunological target to preferentiallytarget tumor cells versus normal cells. In some embodiments, a tumorantigen is a self-antigen.

Tumor antigens include tumor-specific antigens (TSA) andtumor-associated antigens (TAA). Tumor-specific antigens may beexpressed by tumor cells only (generally not expressed on any othercell). Tumor-associated antigens are expressed by tumor cells and bysome normal (non-tumor) cells.

Examples of tumor antigens of the present disclosure include, withoutlimitation, CMV antigens, antigens presented on or within brain cancercells, e.g., glioblastoma, MAGE-1, AIM-2, gp100, and HER2. Otherexamples of tumor antigens of the present disclosure include, withoutlimitation, 9D7, adipophilin, AIM-2, ALDH1A1, alpha-actinin-4,alpha-foetoprotein, ARTC1, BCLX (L), BING-4, B7-H3, B7-H6, BCMA,BCR-ABL, beta-catenin, B-RAF, BAGE family antigens, CAGE familyantigens, Cyclin-A1, CASP-5, CASP-8, CALCA, CD274, CPSF, cyclin-B1,cyclin D1, Cdc27, CDK4, CDK12, CDKN2A, CEA, CLPP, COA-1, CSNK1A1,CD19,CD20, CD21, CD22, CD45, CD70, CSPG4, dek-can, DKK1, ENAH (hMena), EpCAM,EphA3, EZH2, EFTUD2, EGFRvIII, Elongation factor 2, EphA2, EGFR family,ETV6-AML1, FLT3-ITD, FN1, FAP, FRa, glupican-3, FGF5, Ganglioside GD3,glypican-3, G250/MN/CAIX, GD2, GD3, GAGE family antigens, GAS7,glycoprotein 100 (gp100), Gp100/pme117, GPNMB, HAUS3, HER2 (ErbB2),HER-2/neu, HLA-DOB, Hepsin, HERV-K-MEL, HLA-A2, HLA-A11, hsp70-2,HLA-A1+MAGE1, ISO01, IGF2B3, Intestinal carboxyl esterase, IL-11Ra,IL13Ra2, IL-23Ra2, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KM-HN-1,LDLR-fucosyltransferaseAS, LAGE-1, MAGE family antigens, Lengsin,Lewis-Y, MART2, MATN, mammaglobin-A, Melan-A/MART-1, M-CSF, MCSP, mdm-2,ME1, Meloe, mesothelin, Midkine, MMP-2, MMP-7, MUC1, MUC-1, MUC5AC,MUC-16, MUM-1, MUM-2, MUM-3, MART-1, MAGE-A3, Myosin class I, neo-PAP,N-ras, NA88-A, NY-BR-1, NY-ESO-1/LAGE-2, NFYC, NY-ESO-1, OA1, OGT, OS-9,PAP, PAXS, PBF, PSA, p53, pmI-RARalpha, PPP1R3B, PRDXS, PTPRK, PSMA,PRAME, RAB38/NY-MEL-1, RAGE-1, RGSS, RhoC, RNF43, RU2AS, NKG2D ligandsROR1, RBAF600, SAGE family antigens, SAP-1, secernin 1, SIRT2, SNRPD1,SOX10, STEAP1, survivin, SYT-SSX1, SYT-SSX2, Sp17, SSX family antigens,TAG-1, TAG-2, Telomerase, TPBG, TRAG-3, TRP2-INT2, TAG72, TGF-betaRII,Triosephosphate isomerase, TRP-1/gp75, TRP-2, tyrosinase, VEGFR2, WT1,or XAGE family antigens. Other examples of tumor antigens are described(der Bruggen P et al. Peptide database: T cell-defined tumor antigens.Cancer Immun 2013, cancerimmunity.org/peptide, incorporated herein byreference). Other tumor antigens are encompassed by the presentdisclosure.

In some embodiments, other antigens that could be used include but arenot limited to total tumor RNA antigens (antigens derived from totaltumor RNA) and tumor-specific antigens, such as conserved mutations(IDH1, K27M, BRAF V600E mutation, EGFRvIII) (“conserved mutationantigen”) or patient-specific mutations identified by DNA or RNAsequencing or protein analysis (“patient-specific mutation antigen”).

Cancer. The therapies described herein include treatment of an existingor established cancer, that is, one that exists and is detectable in thesubject. Additionally, treatment of a precancerous lesion (e.g.,adenomatous polyp, or cellular dysplasia) for the prevention of thedevelopment of cancer is envisioned. Cancers treatable according to thecurrent disclosure include the following cancers: glioblastoma,melanoma, squamous cell carcinoma, basal cell carcinoma, breast cancer,head and neck carcinoma, thyroid carcinoma, soft tissue sarcoma, bonesarcoma, testicular cancer, prostatic cancer, ovarian cancer, bladdercancer, skin cancer, brain cancer, medulloblastoma, ependymoma,angiosarcoma, hemangiosarcoma, mast cell tumor, primary hepatic cancer,small cell lung cancer, non-small-cell lung cancer, pancreatic cancer,gastrointestinal cancer, renal cell carcinoma, hematopoietic neoplasia,lymphoma, mesothelioma, or a metastatic cancer thereof. In embodimentsof the disclosure, the cancers to be treated in the disclosure includeglioblastoma, low-grade glioma, high-grade glioma, pediatric braincancer, and medulloblastoma. In embodiments of the disclosure, thecancer is invasive intracranial glioma. In embodiments of thedisclosure, the cancer is a metastatic or refractory cancer of thebrain, lung, breast, or melanoma. In embodiments of the disclosure, thecancer is a metastatic brain cancer from non-small cell lung cancer, ametastatic brain cancer from melanoma, or a metastastic brain cancerfrom breast carcinoma.

The present disclosure encompasses the treatment of all tumor types,including primary tumors and metastatic tumors. Tumors that arise fromconnective tissue, endothelium, mesothelium, blood cells, lymphoidcells, muscle, epithelial tissue, neural tissue and neural crest-derivedcells are encompassed herein. The present disclosure also encompassescarcinomas, sarcomas, myelomas, leukemias, lymphomas, and cancers ofmixed type (e.g., adenosquamous, carcinoma, mixed mesodermal tumor,carcinosarcoma and teratocarcinoma).

The following is a list of non-limiting examples of tumors/cancersencompassed by the present disclosure: acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), adrenocortical carcinoma,AIDS-related cancers, Kaposi sarcoma, AIDS-related lymphoma, primary CNSlymphoma, anal cancer, appendix cancer, astrocytomas, atypicalteratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladdercancer, bone cancer, Ewing sarcoma family of tumors, osteosarcoma andmalignant fibrous histiocytoma, brain stem glioma, glioblastoma, braintumor, astrocytomas, brain and spinal cord tumors, brain stem glioma,central nervous system atypical teratoid/rhabdoid tumor, central nervoussystem embryonal tumors, central nervous system germ cell tumors,craniopharyngioma, ependymoma, breast cancer, bronchial tumors, Burkittlymphoma, carcinoid tumor, gastrointestinal, carcinoma of unknownprimary, cardiac (heart) tumors, atypical teratoid/rhabdoid tumor,embryonal tumors, germ cell tumor, primary lymphoma, cervical cancer,cholangiocarcinoma, chordoma, chronic lymphocytic leukemia (CLL),chronic myelogenous leukemia (CML), chronic myeloproliferativeneoplasms, colon cancer, colorectal cancer, craniopharyngioma, cutaneousT-cell lymphoma, ductal carcinoma in situ (DCIS), embryonal tumors,central nervous system, endometrial cancer, ependymoma, esophagealcancer, esthesioneuroblastoma, ewing sarcoma, extracranial germ celltumor, extragonadal germ cell tumor, eye cancer, intraocular melanoma,retinoblastoma, fallopian tube cancer, fibrous histiocytoma of bone,malignant, and osteosarcoma, gallbladder cancer, gastric (stomach)cancer, gastrointestinal carcinoid tumor, gastrointestinal stromaltumors (gist), germ cell tumor, central nervous system, extracranial,extragonadal, ovarian, testicular, gestational trophoblastic disease,glioma, brain stem, hairy cell leukemia, head and neck cancer, heartcancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell,Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, isletcell tumors, pancreatic neuroendocrine tumors, kaposi sarcoma, kidney,renal cell, Wilms tumor and other kidney tumors, langerhans cellhistiocytosis, laryngeal cancer, acute lymphoblastic leukemia (ALL),acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL),chronic myeloid leukemia (CML), hairy cell, lip and oral cavity cancer,liver cancer (primary), lung cancer, non-small cell, small cell,lymphoma, Burkitt, cutaneous t-cell, Hodgkin, non-Hodgkin, primarycentral nervous system (CNS), macroglobulinemia, waldenstrom, malebreast cancer, malignant fibrous histiocytoma of bone and osteosarcoma,melanoma, intraocular (eye), merkel cell carcinoma, mesothelioma,malignant, metastatic squamous neck cancer with occult primary, midlinetract carcinoma involving nut gene, mouth cancer, multiple endocrineneoplasia syndromes, multiple myeloma/plasma cell neoplasm, mycosisfungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferativeneoplasms, myelogenous leukemia, myeloma, myeloproliferative neoplasms,chronic, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer,neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ocular,oral cancer, oral cavity cancer, lip and, oropharyngeal cancer,osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer,epithelial, germ cell tumor, low malignant potential tumor, pancreaticcancer, pancreatic neuroendocrine tumors (islet cell tumors),papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer,parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma,pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonaryblastoma, pregnancy and breast cancer, primary central nervous system(CNS) lymphoma, primary peritoneal cancer, prostate cancer, rectalcancer, renal cell (kidney) cancer, renal pelvis and ureter,transitional cell cancer, retinal cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcoma, ewing, kaposi,osteosarcoma (bone cancer), rhabdomyosarcoma, soft tissue, uterine,Sézary syndrome, skin cancer, melanoma, merkel cell carcinoma,nonmelanoma, small cell lung cancer, small intestine cancer, soft tissuesarcoma, squamous cell carcinoma, squamous neck cancer with occultprimary, metastatic, stomach (gastric) cancer, t-cell lymphoma,cutaneous, testicular cancer, throat cancer, thymoma and thymiccarcinoma, thyroid cancer, transitional cell cancer of the renal pelvisand ureter, unknown primary, carcinoma of, unusual cancers of, ureterand renal pelvis, transitional cell cancer, urethral cancer, uterinecancer, endometrial, uterine sarcoma, vaginal cancer, vulvar cancer andwaldenstrom macroglobulinemia.

Infectious Disease. The disclosure also is useful in connection with thetreatment of infectious disease. In general, an opportunistic pathogenicmicroorganism may be categorized as virus, fungus, parasite, orbacterium. Illustrative pathogenic viral organisms causing humandiseases include (but are not restricted to) Filoviruses, Herpesviruses, Hepatitis viruses, Retroviruses, Human Immunodeficiency Virus(HIV), orthomyxoviruses, Paramyxoviruses, Togaviruses, Picornaviruses,Papovaviruses and Gastroenteritisviruses. Illustrative pathogenicbacteria causing serious human diseases are the Gram positive organisms:Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalisand E. faecium, Streptococcus pneumoniae and the Gram negativeorganisms: Pseudomonas aeruginosa, Burkholdia cepacia, Xanthomonasmaltophila, Escherichia coli, Enterobacter spp, Klebsiella pneumoniaeand Salmonella spp. Illustrative pathogenic protozoan organisms causinghuman diseases include (but are not restricted to) Malaria e.g.,Plasmodium falciparum and M. ovale, Trypanosomiasis (sleeping sickness)e.g., Trypanosoma cruzei, Leischmaniasis e.g., Leischmania donovani,Amebiasis e.g., Entamoeba histolytica. Illustrative pathogenic fungicausing or associated with human diseases include (but are notrestricted to) Candida albicans, Histoplasma neoformans, Coccidioidesimmitis and Penicillium marneffei. In some embodiments, the infectiousdisease organism is one involved in chronic infectious disease.Particularly important diseases are hepatitis, adenovirus, polyoma virussuch as BK, human immunodeficiency virus (HIV), herpes simplex virus(HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV),Epstein-Barr virus (EBV), Influenza A, B, and C, vesicular stomatitisvirus (VSV), vesicular stomatitis virus (VSV), Staphylococcus speciesincluding Methicillin-resistant Staphylococcus aureus (MRSA),Streptococcus species including Streptococcus pneumonia, andpost-transplant infection.

Cytomegalovirus (CMV) and CMV antigens. Cytomegalovirus (CMV) is a genusof viruses in the order Herpesvirales, in the family Herpesviridae, inthe subfamily Betaherpesvirinae. There are currently eight species inthis genus including the type species human herpesvirus 5. Human CMV(HCMV) or human herpesvirus-5 (HHV-5) is a species that infects humansand is the most studied of all cytomegaloviruses. Diseases associatedwith HHV-5 include mononucleosis, and pneumonias. CMV antigens are knownin the art and include CMV early and late membrane antigens (CMV-EMA andCMV-LMA, respectively). In some embodiments, CMV antigens are used inthe compositions and methods.

Autoimmune disease. The disclosure also is useful in connection with thetreatment of autoimmune disease. In some embodiments, to treatautoimmune disease, APCs may be generated to be tolerogenic rather thanimmunogenic when administered to the host. In some embodiments,tolerogenic dendritic cells may be generated and used. The cells may beadministered to the host to induce T cell tolerance and/or deletionrather than activation. Autoimmune diseases include, but are not limitedto rheumatoid arthritis, Crohn's disease or colitis, multiple sclerosis,systemic lupus erythematosus (SLE), autoimmune encephalomyelitis,myasthenia gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome,pemphigus (e.g., pemphigus vulgaris), Grave's disease, autoimmunehemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma withanti-collagen antibodies, mixed connective tissue disease, polymyositis,pernicious anemia, idiopathic Addison's disease, autoimmune-associatedinfertility, glomerulonephritis (e.g., crescentic glomerulonephritis,proliferative glomerulonephritis), bullous pemphigoid, Sjogren'ssyndrome, insulin resistance, and autoimmune diabetes mellitus (type 1diabetes mellitus; insulin-dependent diabetes mellitus). Autoimmunedisease has been recognized also to encompass atherosclerosis andAlzheimer's disease. In some embodiments of the aspects describedherein, the autoimmune disease is selected from the group consisting ofmultiple sclerosis, type-I diabetes, Hashimoto's thyroiditis, Crohn'sdisease or colitis, rheumatoid arthritis, systemic lupus erythematosus,gastritis, autoimmune hepatitis, hemolytic anemia, autoimmunehemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmuneuveoretinitis, glomerulonephritis, Guillain-Barre syndrome, psoriasisand myasthenia gravis.

Other diseases that may be treated with this technology include glioma,medulloblastoma, ependymoma, primitive neuroectodermal tumors, brainstem gliomas, other primary brain tumors, solid tissue cancer with orwithout metastasis, non-cancerous tumors, hematologic malignancy, andautoimmune disorders. Antigens that could be used include but are notlimited to CMV antigens, total tumor RNA, and tumor-specific antigenssuch as conserved mutations (IDH1, K27M, BRAF V600E mutation, EGFRvIII)or patient-specific mutations identified by DNA or RNA sequencing orprotein analysis.

Methods of Administration. In some embodiments, the cell compositionsare administered intravenously (by intravenous (IV) infusion). In someembodiments, the cell compositions described herein, tetanus/diphtheriatoxoid, or both, are delivered to a subject via a parenteral route or anenteral route. In some embodiments, the cell compositions describedherein, tetanus/diphtheria toxoid, or both, are delivered to a subjectvia intradermal or intravenous injection. In some embodiments, the cellcomposition described herein is administered to the subject immediatelyafter the cell is cultured in and/or electroporated with sarcosine. Insome embodiments, the cell composition described herein is administeredto the subject via intradermal or intravenous injection immediatelyafter the cell is cultured in and/or electroporated with sarcosine.Examples of parental routes include, without limitation,intra-abdominal, intra-amniotic, intra-arterial, intra-articular,intrabiliary, intrabronchial, intrabursal, intracardiac,intracartilaginous, intracaudal, intracavernous, intracavitary,intracerebral, intracisternal, intracorneal, intracoronal,intracoronary, intracorporus, intracranial, intradermal, intradiscal,intraductal, intraduodenal, intradural, intraepidermal, intraesophageal,intragastric, intragingival, intraileal, intralesional, intraluminal,intralymphatic, intramedullary, intrameningeal, intramuscular,intranasal, intraocular, intraovarian, intrapericardial,intraperitoneal, intrapleural, intraprostatic, intrapulmonary,intraocular, intrasinal, intraspinal, intrasynovial, intratendinous,intratesticular, intrathecal, intrathoracic, intratubular,intratympanic, intrauterine, intravascular, intravenous (including bolusor drip), intraventricular, intravesical and subcutaneous.

Enteral routes of administration include, without limitation,administration to the gastrointestinal tract via the mouth (oral),stomach (gastric) and rectum (rectal). Gastric administration typicallyinvolves the use of a tube through the nasal passage (NG tube) or a tubein the belly leading directly to the stomach (PEG tube). Rectaladministration typically involves rectal suppositories.

In some embodiments, compositions are delivered to a subject prior to orafter treatment with a tetanus/diphtheria toxoid. Thus, the compositionsdescribed herein and tetanus/diphtheria toxoid may be deliveredsequentially or simultaneously. Hematopoietic Stem Cell. A hematopoieticstem cell (HSC), also called a blood stem cell, is an immature cellfound in the blood and the bone marrow that can renew itself, and thatcan differentiate into a variety of specialized cells, such as blood andimmune cells, including white blood cells, red blood cells, andplatelets. HSCs can mobilize out of the bone marrow into circulatingblood. HSCs facilitate constant renewal of blood cells, producingbillions of new blood cells each day.

Hematopoietic Stem Cell Transplantation (HSCT). Hematopoietic stem celltransplantation (HSCT) is the transplantation of HSCs, usually derivedfrom peripheral blood, bone marrow, or umbilical cord blood. Two typesof HSCT may be used in a subject: autologous stem cell transplantation,wherein the subject's own stem cells are used, or allogenic stem celltransplantation, wherein a donor's stem cells, that are geneticallysimilar and HLA-matched to the recipient, are transplanted into thesubject. In some embodiments of the disclosure, autologous stem cellsare used for HSCT.

In autologous HSCT, a sample containing stem cells are removed from thesubject, stored, and later transplanted back into the subject. HSCsrepresent a small fraction of the total population of blood cells in thesample, so it may be advantageous to increase the number of HSCs beforeadministering them to the subject for cancer or infectious diseasetherapy. In some embodiments, hematopoietic stem cells are collected andexpanded, before transplanting them into the subject for treatment. Insome embodiments, hematopoietic stem cells are collected, expanded, andselected for from the sample, before transplanting them into the subjectfor treatment.

In some embodiments, stem cells can be enriched in the material used fortransplantation. In some embodiments, the enrichment can occur byselectively stimulating the growth/expansion of stem cells versus othercells collected from a subject. In other embodiments, the stem cells canbe enriched by isolating stem cells from other cells collected from asubject. Such selection may be so-called positive selection or negativeselection. In positive selection, stem cells are isolated based onmarkers known to be on stem cells but not on other cells. In negativeselection, cells that are not stem cells are identified and removedbased on markers on such other cells, leaving behind stem cells. Suchprocedures are well known to those of ordinary skill in the art.

Sources of hematopoietic stem cells herein include: bone marrow lineagedepleted cells (lin−), cKit+ purified lineage negative bone marrowderived cells, Sca+ purified lineage negative bone marrow derived cells,cKit+Sca+ purified bone marrow derived cells, mobilized from host bonemarrow using G-CSF, mobilized from host bone marrow using AMD3100,Plerixafor, or the molecule1,1′-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane],umbilical cord blood or cord-blood derived stem cells, human leukocyteantigen (HLA)-matched blood, mesenchymal stem cells derived from bloodor marrow, hematopoietic stem cells differentiated from inducedpluripotent stem cells, mobilized peripheral blood, peripheral blood,hematopoietic stem cell subsets including Lin− cells purified with CCR2+marker, lineage negative purified peripheral blood, or CD34+ enrichedperipheral blood. In some embodiments of the disclosure, the source ofHSCs is bone marrow. In some embodiments of the disclosure, the sourceof HSCs is autologous or allogeneic, optionally wherein, the source isbone marrow, peripheral blood, umbilical cord blood, or inducedpluripotent stem cells.

In some embodiments, HSCs (autologous or allogeneic) are provided withor loaded with sarcosine in superphysiological (above physiological)amounts. In some embodiments, HSCs provided or loaded with sarcosineabove physiological amounts are administered to a subject. In someembodiments, HSCs provided or loaded with sarcosine above physiologicalamounts are administered to a subject in combination with anothertreatment or therapy.

Subject. “Subject” means a mammal, such as a human, a nonhuman primate,a dog, a cat, a sheep, a horse, a cow, a pig, a mouse, a rat, a rodent,or a goat. In an important embodiment, the subject and/or mammal is ahuman.

Treatment. “Treat”, “treating”, “treatment”, and “therapy” encompass anaction that occurs while a subject is suffering from a condition whichreduces the severity of the condition (or a symptom associated with thecondition) or retards or slows the progression of the condition (or asymptom associated with the condition). This is therapeutic treatment.

Effective Amount. Subjects are treated with effective amounts of thecell compositions of the disclosure. An “effective amount” of acomposition or an agent generally refers to an amount sufficient toelicit the desired biological response, i.e., treat the condition. Aswill be appreciated by those of ordinary skill in the art, the effectiveamount of a composition described herein may vary depending on suchfactors as the condition being treated, the mode of administration, andthe age, body composition, and health of the subject.

For therapeutic treatment, an effective amount is an amount sufficientto provide a therapeutic benefit in the treatment of a condition or toreduce or eliminate one or more symptoms associated with the condition.This may encompass an amount that improves overall therapy, reduces oravoids symptoms or causes of the condition, or enhances the therapeuticefficacy of another therapeutic agent.

In one aspect, effective amounts of cells administered are those amountssufficient to enhance an immune response in the subject. In connectionwith a specific disease or condition, “enhance an immune response” meansto halt the development of, inhibit the progression of, reverse thedevelopment of, or otherwise reduce or ameliorate one or more symptomsof the disease or condition, for example, one or more symptoms of canceror one or more symptoms of an infectious disease. In addition, effectiveamounts may be such amounts which slow, halt or reverse the growth ofcancer cells/tumors or an infectious disease in the subject. Inaddition, effective amounts may be such amounts which improve asubject's immune response to an antigen.

In another aspect, effective amounts are those amounts of sarcosinesufficient to enhance targeting cells to lymph nodes.

Adoptive Cell Therapy (ACT or adoptive cell transfer). Adoptive celltherapy is the transfer of cells into a patient for the purpose oftransferring immune functionality and other characteristics with thecells. The cells are most commonly immune-derived, for example T cells,and can be autologous or allogeneic. Transfer of autologous cells ratherthan allogeneic cells minimizes graft versus host disease issues. ACTcan be used for treatment of viral infections and/or cancer. There is anincreased risk for infection and/or malignancy, in a subject receivingimmunosuppressive or ablative treatment (e.g., chemo or radiationtreatment) in connection, for example, with stem cell transplantation,including HSCT, organ transplantation, and certain types of cancer,wherein immune reconstitution is often slow and incomplete and there isa risk for malignancy. The use of ACT in a subject in the periodfollowing immunosuppression is thought to be advantageous to thesubject, with the potential for enhancing immunity, including antitumorimmunity, and increasing vaccine efficacy in the period followingimmunosuppression. ACT of tumor-specific T cells has been shown to beeffective in treatment of solid tumors in murine and in human systems.In some embodiments, cells used in ACT, e.g. T cells (cytotoxic T cells,T helper cells) can be provided with sarcosine in superphysiologicalamounts through culture medium and/or electroporation.

EXAMPLES Example 1. Loading cells with Sarcosine

Fresh 1 molar sarcosine (Sigma) solution was prepared by dissolvingsarcosine in OptiMEM medium (medium used for electroporation). Cellsharvested on day eight of DC culture were counted and re-suspended at adensity of about 5000K/200 μl in either OptiMEM only (control), 6millimolar (mM), or 0.5 Molar (M) sarcosine solution in OptiMEM andelectroporated at 300V for 500 microseconds. Immediately afterelectroporation, the cells were resuspended in 5 ml of complete DCmedium with IL-4 and GM-CSF and incubated overnight at 37° C. ControlDCs were plated in culture medium only and the 6 mM and 0.5 M sarcosineelectroporated DCs were plated in culture medium containing 6 mM, or 20mM of sarcosine, respectively.

FIG. 1 shows amounts (pg) of sarcosine per cell under differenttreatment conditions. Column 1 shows the physiological amount ofsarcosine per cell. Column 2 shows the amount of sarcosine per cell inDCs electroporated with 6 mM sarcosine and cultured in 6 mM sarcosinefor 24 hours at 37° C. Column 3 shows the amount of sarcosine per cellin DCs electroporated with 0.5 M sarcosine and cultured in 20 mMsarcosine for 24 hours at 37° C. Electroporation was performed for 0.5seconds at 300V, after which the cells were resuspended in mediumcontaining 6 mM or 20 mM sarcosine and cultured overnight at 37° C.Cells electroporated with 0.5 M sarcosine and cultured in 20 mMsarcosine containing medium yield the highest amount of intracellularsarcosine. In some embodiments, the cell is cultured in sarcosine. Insome embodiments, the cell is cultured in sarcosine and electroporatedwith sarcosine. Sarcosine in the culture may range between 0 M and about1 M sarcosine, e.g., 0.0 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M,0.7 M, 0.8 M, 0.9 M, or 1 M sarcosine, without electroporation orbetween about 6 mM and about 20 mM sarcosine, e.g., 6 mM, 7 mM, 8 mM, 9mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19mM, or 20 mM, with electroporation.

Example 2. Measuring Levels/Amounts of Sarcosine

Standard methods known in the art may be used to measure sarcosine incells. For example see Sarcosine assay kit (Sigma, MAK073-1.KT,incorporated by reference, herein). This kit was used for measuringamounts of sarcosine per cell, as in FIG. 1.

Example 3. In Vitro Study to Measure Migration of Dendritic Cells (DCs)Under Different Conditions

DCs harvested from different conditions (sarcosine vs no-sarcosine) wereresuspended in RPMI medium plus 0.2% fetal bovine serum (FBS) at 1×10⁶DC/mL. To evaluate the migratory capacities of DCs, a 12 well-Transwellmicroplate (Corning) with a 5 μm membrane pore size was used. For eachcondition tested, lower chambers of the Transwell were filled with 600μL RPMI medium supplemented with 0.2% fetal bovine serum (FBS) with orwithout 250 ng/mL CCL19 and CCL21. 100 μL of DCs (1×10⁵) were pipettedinto the upper chamber of the Transwell and then the plated cells wereincubated for 3 hours at 37° C. Migrated DCs were harvested from thelower chamber and were counted using a hemocytometer (FIG. 2). Cytokinesare a chemoattractant agent for the cells. Cytokines used were CCL-19and CCL-21. Dendritic cells (no sarcosine treatment, no cytokines added)show a baseline amount of migration (column 1). Dendritic cellselectroporated with 6 mM sarcosine and then cultured in 6 mM sarcosinecontaining medium overnight (column 2) show a very small difference inmigration from DCs alone. Migration of DCs (not treated with sarcosine)increases with addition of cytokines (column 3). Migration of sarcosineloaded DCs with addition of cytokines (column 4) is increased even moreas compared to DCs with addition of cytokines (but not loaded orprovided with sarcosine).

Example 4. In Vivo Study to Assess Migration of Cells to Local LymphNode

Mice were vaccinated with DCs under four different vaccine conditionsand the percentage of total cells that migrated to the local lymph nodewas measured (FIG. 3). Briefly, animals were randomized into Td(tetanus/diphtheria) primed and control groups. Protocols forpre-conditioning or priming with tetanus toxoid (Td) in mice and humansare provided in Mitchell et al., Nature Letter (2015) 519:19, includingsupplement, incorporated herein by reference, in its entirety. Eachgroup of animals was then divided into two subgroups depending on thetype of DC vaccine they received: DC only or sarcosine loaded DCs. Fortyeight (48) hours after receiving intradermal DC vaccination, thedraining lymph nodes from each animal were carefully resected andprocessed to obtain single cell suspension. Knowing the number of cellsharvested from each animal and the percentage of PKH DCs that migratedto the lymph node, the percentage of vaccinated DCs that migrated to thelymph node was calculated. The percentage of PKH DCs that migrated tothe local lymph node was measured by flow cytometry (FIG. 3). In controlmice with no Td vaccine, less than 10% of DCs (control DCs or sarcosineloaded DCs) used for vaccination migrated to the local lymph node(columns 1, 2). In mice primed with Td vaccine, around 20% of controlDCs (no sarcosine) and around 30% of sarcosine loaded DCs migrated tothe local lymph node (columns 3, 4). When tetanus toxoid vaccinationpriming was combined with vaccination with sarcosine-loaded DCs, asynergistic effect on enhanced migration was observed (column 4).

Example 5. NMR of Sarcosine Loaded DCs

Sarcosine has a unique spectral peak that can be identified with NMR.FIG. 4 shows sarcosine content of pure and diluted sarcosine loaded DCsanalyzed 24 hours after cells were loaded with sarcosine. Cells wereelectroporated in 0.5 M sarcosine solution and then cultured for 24hours in 20 mM of sarcosine. The cells were analyzed for sarcosinedetection using NMR and they were serially diluted with unlabeled DCs.Loaded DCs can be traced to 5% dilution. Results shown in FIG. 4 supportthe hypothesis that MR spectroscopy can be used to identify sarcosineloaded cells that migrate to draining lymph nodes.

Example 6. SARDH siRNA Treatment

Dendritic cells were cultured in media containing 20 mM sarcosine asdescribed in Example 1. Cells were then electroporated at 300V with 0.5mM sarcosine, or electroporated with siRNA against SARDH (sarcosinedehydrogenase) to reduce metabolism of sarcosine. The cells wereanalyzed using the Sarcosine kit as described in Example 2.

FIG. 5 shows the amounts (pg) per cell under different treatmentconditions. Column 1 shows the physiological amount of sarcosine percell. Column 2 shows the amount of sarcosine per cell in DCs cultured in20 mM sarcosine. Column 3 shows the amount of sarcosine per cell in DCscultured in 20 mM sarcosine and electroporated with 0.5 mM sarcosine.Column 4 shows the amount of sarcosine per cell in DCs cultured in 20 mMsarcosine and electroporated with siRNA against SARDH.

The results presented herein show that neither electroporation with 0.5mM sarcosine nor electroporation with siRNA against SARDH increased theamount of sarcosine per cell above that of cells cultured in 20 mMsarcosine.

The cultured dendritic cells were also examined by flow cytometry todetermine wither the culture conditions changed their DC phenotype. Thephenotype expected was CD80, CD86, CD11c, and IAb positive and Gr1negative. The cultured cells were incubated with antibodies for CD80,CD86, CD11c, GR1 and IAb and flow cytometry was performed to evaluatethe DC phenotype. Sarcosine loaded cells maintained a DC phenotype whencompared to control DCs.

Example 7. In Vivo Study to Assess Migration of Cells to Lymph Nodes andSpleen

Dendritic cells were cultured and loaded with sarcosine as described inExample 1. DCs were also loaded with PKH cell membrane dye. Sarcosineloaded or control DCs were given as an intradermal injection to C57BL/6mice. After 48 hours, the lymph nodes and spleens were collected fromthe mice and immunofluorescence was performed to visualize PKH+ cells(which are the injected DCs) and B220+ cells (which are B cells). Theanimals that received sarcosine labeled DCs had more migration to thelymph nodes and the spleens at 48 hours post vaccination compared toanimals receiving control DCs. There was vastly more migration to thelymph nodes/spleen. It appeared on visual inspection that sarcosineincreased migration to the lymph nodes and the spleen more than twotimes, three times, four times more, five times and perhaps even morethan ten-fold more than controls.

1. A composition comprising an antigen presenting cell (APC), whereinthe cell comprises a receptor for presenting antigen and antigen boundto the receptor, and wherein the cell contains at least 0.1 pg ofsarcosine.
 2. A composition comprising an antigen presenting cell (APC),wherein the cell comprises a receptor for presenting antigen and antigenbound to the receptor, and wherein the APC is cultured in the presenceof media containing sarcosine and/or the APC is electroporated withsarcosine.
 3. A cell culture composition comprising: liquid mediumcontaining sarcosine in concentrations above physiological levels, andan antigen presenting cell (APC).
 4. The composition of claim 3, whereinthe APC has a receptor for presenting antigen and antigen bound to thereceptor.
 5. The composition of claim 2, wherein the sarcosine is abovephysiological levels in the cell.
 6. The composition of claim 1, whereinthe cell contains between about 0.1 and 15 pg of sarcosine.
 7. Thecomposition of claim 2, wherein the cell contains at least 0.1 pg ofsarcosine.
 8. The composition of claim 1, wherein the APC is a humancell.
 9. The composition of claim 1, wherein the APC is a dendritic cell(DC).
 10. The composition of claim 1, wherein the antigen is adisease-associated antigen.
 11. The composition of claim 10, wherein theantigen is a human cytomegalovirus (CMV) antigen.
 12. The composition ofclaim 10, wherein the disease-associated antigen is a tumor antigen. 13.The composition of claim 10, wherein the antigen is from total tumorRNA.
 14. The composition of claim 10, wherein the antigen is a conservedmutation antigen or a patient-specific mutation antigen.
 15. Thecomposition of claim 10, wherein the tumor antigen is an antigenpresented on or within brain cancer cells.
 16. The composition of claim12, wherein the tumor antigen is a brain tumor antigen.
 17. Thecomposition of claim 16, wherein the brain tumor antigen is aglioblastoma antigen.
 18. The composition of claim 10, wherein thedisease-associated antigen is an infectious disease antigen.
 19. Thecomposition of claim 1, wherein the antigen is presented to the APC by:pulsing the APC; fusing cell components with disease tissue components;viral gene transduction; or DNA transfer.
 20. The composition of claim1, wherein the antigen is a short peptide, a long peptide, mRNA, DNA,recombinant protein, tumor lysate mixture, or carbohydrate antigenexpressed within a cancer cell or infected cell.
 21. A method forpreparing a cell-based pharmaceutical preparation comprising: providingan antigen presenting cell comprising a receptor for presenting antigenand contacting the cell with a solution containing sarcosine.
 22. Amethod of treating a disease in a subject, comprising: administering tothe subject an antigen presenting cell (APC), wherein the cell comprisesa receptor for presenting antigen and antigen bound to the receptor andwherein the cell contains at least 0.1 pg of sarcosine. 23-40.(canceled)